A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.     

Thymoquinone suppression of the human hepatocellular carcinoma cell growth involves inhibition of IL-8 expression,elevated levels of TRAIL receptors,oxidative stress and apoptosis

Sod2 is the major salt tolerance plasma membrane protein of Schizosaccharomyces pombe. It functions to remove excess intracellular sodium (or lithium) in exchange for protons. We investigated the role of cysteine residues and created a cysteine-free Sod2 protein. Each cysteine residue of the ten present was individually mutated to serine and the different proteins expressed and characterized in S. pombe. Western blotting revealed that all the individual mutant proteins were expressed. We examined the ability of the mutant proteins to confer salt tolerance to S. pombe with the endogenous Sod2 protein deleted. Only proteins with C26S and C374S mutations were partially reduced in their ability to confer salt tolerance. Additionally, they showed a change in conformation in comparison to the wild-type protein, indicated by differential sensitivity to trypsin. Deletion of all the cysteine residues of Sod2 resulted in a functional protein that was expressed in S. pombe at levels similar to the wild type and also conferred salt tolerance. The conformation of the cysteine-free Sod2 protein was not altered relative to the wild-type protein. We examined the accessibility of amino acids of the cysteineless protein present on putative extracellular loop 2. A cysteine placed at position Ala119 was accessible to externally applied [2-(trimethylammonium)ethyl] methane thiosulfonate bromide. The results demonstrate that cysteines in the Sod2 protein can be changed to serine residues resulting in an expressed, functional protein. The utility of the cysteine-free Sod2 protein for determination of topology and amino acid accessibility is demonstrated.     

Molecular cloning and characterization of genistein 4′-O-glucoside specific glycosyltransferase from Bacopa monniera

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4′-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4′-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49 % identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC–MS and HPLC, which confirmed its specificity for genistein 4′-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.     

ECHIDNA Protein Impacts on Male Fertility in Arabidopsis by Mediating trans-Golgi Network Secretory Trafficking during Anther and Pollen Development

The trans-Golgi network (TGN) plays a central role in cellular secretion and has been implicated in sorting cargo destined for the plasma membrane. Previously, the Arabidopsis (Arabidopsis thaliana) echidna (ech) mutant was shown to exhibit a dwarf phenotype due to impaired cell expansion. However, ech also has a previously uncharacterized phenotype of reduced male fertility. This semisterility is due to decreased anther size and reduced amounts of pollen but also to decreased pollen viability, impaired anther opening, and pollen tube growth. An ECH translational fusion (ECHPro:ECH-YELLOW FLUORESCENT PROTEIN) revealed developmentally regulated tissue-specific expression, with expression in the tapetum during early anther development and microspore release and subsequent expression in the pollen, pollen tube, and stylar tissues. Pollen viability and production, along with germination and pollen tube growth, were all impaired. The ech anther endothecium secondary wall thickening also appeared reduced and disorganized, resulting in incomplete anther opening. This did not appear to be due to anther secondary thickening regulatory genes but perhaps to altered secretion of wall materials through the TGN as a consequence of the absence of the ECH protein. ECH expression is critical for a variety of aspects of male reproduction, including the production of functional pollen grains, their effective release, germination, and tube formation. These stages of pollen development are fundamentally influenced by TGN trafficking of hormones and wall components. Overall, this suggests that the fertility defect is multifaceted, with the TGN trafficking playing a significant role in the process of both pollen formation and subsequent fertilization.Pollen production and release is a critical stage in plant development that typically involves gene expression from over half of the genome. The extent of genomic involvement in pollen development is illustrated by the high frequency of mutations that result in a failure of male fertility; these can be a consequence of the failure of pollen development or pollen release, dehiscence. Detailed analysis of male-sterile mutants in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) has improved the basic understanding of pollen and anther development (Scott et al., 2004; Ma, 2005; Wilson and Zhang, 2009; Cui et al., 2012); however, there are multiple aspects of pollen formation that are still unclear, and many defects result in uncharacterized effects of reduced fertility or complete sterility.The ECHIDNA (ECH) gene was initially identified from expression profiling of the vascular cambium in poplar (Populus spp.) and associated with secondary xylem formation (Hertzberg et al., 2001). The Arabidopsis ech mutant was shown to have a bushy stature with defects in root and hypocotyl elongation, which was linked to defective cell expansion and elongation (Gendre et al., 2011). Analysis of roots in the ech mutant and complementation analyses in yeast showed that the ECH protein impacts on cell expansion by mediating trans-Golgi network (TGN) secretory trafficking but does not affect endocytosis (Gendre et al., 2011). However, in addition to the defects associated with plant stature, the ech mutant also displays a previously unreported phenotype of reduced fertility.Pollen development occurs in a specialized organ, the stamen, which comprises anthers that hold the developing pollen supported by a filament containing the vasculature connections. Stamen primordia arise from divisions in the L1, L2, and L3 layers in the floral meristem. Divisions in the L2 layer result in four clusters of archesporial cells that subsequently form the central sporogenous cells, which are surrounded by four maternal cell layers: the tapetum, middle cell layer, endothecium, and outer epidermis (Scott et al., 2004). The structure of the maternal anther cell layers has been shown to be critical for the production and release of functional pollen, as demonstrated in a number of male-sterile mutants, which have defects in cell division and early stages of differentiation of the tapetum and sporogenous cells. For example, mutants of the Leu-rich repeat receptor kinase EXTRA SPOROGENOUS CELLS (EXS)/EXCESS MICROSPOROCYTES1 (Canales et al., 2002; Zhao et al., 2002) and its ligand TAPETAL DETERMINANT1 (Jia et al., 2008) result in sterility due to the formation of additional male sporocytes and a lack of tapetal cells.The tapetum has been shown to be critical for functional pollen formation, with many of the characterized male-sterile mutants exhibiting abnormal tapetal development, including DYSFUNCTIONAL TAPETUM1 (DYT1; Zhang et al., 2006; Zhu et al., 2008), TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1; Zhu et al., 2008), ABORTED MICROSPORES (AMS; Sorensen et al., 2003; Xu et al., 2010), and MALE STERILITY1 (MS1; Wilson et al., 2001; Ito and Shinozaki, 2002). After differentiation, the tapetum layer becomes metabolically highly active and plays an essential role in the biosynthesis and secretion of sporopollenin and other wall materials for the developing pollen, prior to breakdown via programmed cell death (Ariizumi and Toriyama, 2011). A frequently observed phenotype in male-sterile mutants is enlarged tapetal cells that show defects in secretion and subsequent alterations in programmed cell death breakdown (Wilson and Zhang, 2009). This indicates the important role that the tapetum plays in the regulation of pollen development and, in particular, the passage of materials to the central locule for viable pollen production.Male-sterile phenotypes have also been identified due to a failure of pollen release, dehiscence. Secondary thickening occurs specifically in the endothecium layer of the anther; this layer and the presence of selective thickening within it are critical to generate the differential forces that are required for anther dehiscence and pollen release (Wilson et al., 2011; Nelson et al., 2012). The importance of this secondary thickening is demonstrated in the myb26 mutant (Yang et al., 2007) and in the double NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (nst1 nst2) mutant (Mitsuda et al., 2005), which lack endothecium thickening and, as a result, fail to dehisce (Nelson et al., 2012).Previous investigations of the ech mutation indicated that it is impaired in TGN secretion, resulting in dwarf plants with defects in root and hypocotyl cell elongation. The ech mutant also has an uncharacterized phenotype of impaired male fertility; therefore, a detailed analysis of reproduction in the ech mutant was conducted. ECH expression was seen in the anther tapetum during the early stages of tapetal development and microspore release but was subsequently detected in the pollen, pollen tube, and stylar tissues. The reduced fertility was linked to decreased anther size and pollen production but also to reductions in pollen viability, anther opening, and pollen tube growth. The anther wall thickening was reduced and disorganized in ech, possibly as a consequence of altered secretion of wall materials through the TGN. The male-sterile myb26 mutant has defects in anther endothecium wall thickening resulting in a failure of dehiscence; the ech myb26 double mutant exhibits the phenotypes of both mutants and fails to produce secondary thickening, indicating that the ECH-mediated pathway is acting independently of or upstream through MYB26, possibly by providing the components required for secondary cell wall thickening. The reduction in male fertility, therefore, is likely to be a consequence of multiple effects due to altered secretion in the anther because of impaired TGN transport in the ech mutant; the resulting defects are associated with tapetum and pollen wall development but also anther dehiscence and pollen tube formation.     

Lucrative pectinase production by novel strain Pseudozyma sp. SPJ with statistical optimization techniques using agro-industrial residues

Production of high titers of an alkaline, extracellular and thermo-tolerant pectinase by a newly isolated yeast Pseudozyma sp. SPJ was carried out under solid state fermentation. Citrus peel, the inexpensive agro-industrial residue used as substrate, was experienced to be unsurpassed. Response surface methodology was conducted to optimize the culture conditions for Pseudozyma sp. SPJ for hyper production of pectinase. Plackett Burman design was applied to identify the most effective culture variables. Out of nine variables studied, incubation time, moisture content and ammonium sulfate were detected as most important. A full factorial Central Composite Design was used to optimize the levels of these variables, which resulted in 17-fold increase （71.19 IU/g to 1215.66 IU/g dry substrate） in the enzyme yield. The results of analysis of variance and multiple regression analysis implies that the effect of incubation time （p 〈 0.000） and moisture content （p 〈 0.018） is more than ammonium sulfate. And also the interaction of moisture content with ammonium sulfate （p 〈 0.002） is more significant.     

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background

Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear.

Principle Finding

In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation.

Significance

Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.     

Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila

Modulation of the immune responses using active bio-ingredients as a possible prophylaxis measure has been novel prospect for aquaculture. The present study evaluated the effects of azadirachtin EC 25% on non-specific immune responses in goldfish Carassius auratus and resistance against pathogenic bacteria Aeromonas hydrophila. The experimental trial for effects of azadirachtin on immuno-haematoloical parameters in goldfish was conducted by feeding the various levels of azadirachtin as control T₀ (without azadirachtin), T₁ (0.1%), T₂ (0.2%), T₃ (0.4%), T₄ (0.8%) and T₅ (1.6%) for a period of 28 days. Fishes were challenged with A. hydrophila 28 days post feeding and relative percentage survival (%) was recorded over 14 days post infection. Immuno-haematoloical (total erythrocyte count, total leukocyte count, haemoglobin, packed cell volume, NBT activity, phagocytic activity, serum lysozyme activity, myeloperoxidase activity, total immunoglobulin) and serum biochemical parameters (serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and blood glucose) of fishes were examined at 14 and 28 days of feedings. Fish fed with azadirachtin, showed significantly (p < 0.05) enhanced TEC, TLC, Total Ig, total protein, NBT activity, serum lysozyme activity and myeloperoxidase level in different treatment groups in comparison with control group. Similarly, SGOT, SGPT and blood glucose level were found to be significantly (p < 0.05) high but PCV and Hb did not differ significantly (p > 0.05) in the treatment groups compared to control groups. Azadirachtin at the concentration of 4 g kg^?1 showed significantly (p < 0.05) higher relative percentage survival (42.60%) when compared with the control against A. hydrophila infection. This study indicated that azadirachtin EC 25% (4 g kg^?1) showed higher NBT activity, serum lysozyme, protein profiles, leukocyte counts and resistance against A. hydrophila infection and thus, can be used as a potential immunostimulant in aquaculture.     

Nitroreductase-activated nitric oxide (NO) prodrugs

Due to the involvement of nitric oxide (NO) in numerous and diverse physiological processes, site-directed delivery of therapeutic NO in order to minimize unwanted side-effects is necessary. O²-(4-Nitrobenzyl) diazeniumdiolates are designed as substrates for Escherichia coli nitroreductase (NTR), an enzyme that is frequently used to facilitate directed delivery of cytotoxic species to cancers. O²-(4-Nitrobenzyl) diazeniumdiolates are found to be stable in aqueous buffer but are metabolized by NTR to produce NO. A cell viability assay revealed that cytotoxic effects of O²-(4-nitrobenzyl)1-(2-methylpiperidin-1-yl)diazen-1-ium-1,2-diolate (4b) towards two cancer cell lines is significantly enhanced in the presence of NTR suggesting the potential for use of this compound in nitric oxide-based directed prodrug therapy.